This article presents newly developed screening and confirmation analytical procedures to detect the misuse of nine prolyl-hydroxylase inhibitors of the hypoxia-inducible factor: daprodustat, desidustat, FG2216, IOX2, IOX4, JNJ-42041935, molidustat, roxadustat and vadadustat, targeting either the parent drugs and/or their main metabolite(s). For the sample pre-treatment, different extraction protocols and technologies were evaluated. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. The chromatographic separation was performed on a C18 column, employing water and acetonitrile, both containing 0.1% formic acid, as mobile phase. Detection was achieved using as analyzer either a triple quadrupole or an Orbitrap, with positive and negative electrospray ionization and different acquisition modes. Validation of the procedures was performed according to the ISO 17025 and World Anti-Doping Agency guidelines. The methods do not show any significant interference at the retention times of the analytes of interest. The extraction efficiency was estimated to be greater than 75% for all analytes, and the matrix effect smaller than 35%. Detection capability was determined in the range of 0.25-2.0 for the screening procedure and in the range of 0.5-2.0 ng/mL for the confirmation procedure, that is, in a range of concentration small enough to reveal the abuse of the compounds considered, in case they are used as performance enhancing agents. The repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the selected ion transitions, considered only in the case of triple quadrupole (CV% < 15) was confirmed to be fit for purpose to ensure the unambiguous identification of all the target analytes in human urine. The applicability of the newly developed methods was verified by the analysis of urine samples containing molidustat, roxadustat or daprodustat. The developed procedures enabled to detect the compounds under investigation and their main metabolites.

中文翻译：

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基于 UPLC-MS 的检测尿液中 HIF 脯氨酰羟化酶抑制剂的程序。

具有正负电喷雾电离和不同的采集模式。根据 ISO 17025 和世界反兴奋剂机构指南对程序进行验证。这些方法在目标分析物的保留时间上没有显示出任何明显的干扰。估计所有分析物的提取效率大于 75%，基质效应小于 35%。筛选程序的检测能力在 0.25-2.0 范围内确定，而确认程序的检测能力范围为 0.5-2.0 ng/mL，也就是说，浓度范围小到足以揭示所考虑的化合物的滥用，如果它们被用作性能增强剂。相对保留时间 (CV% < 0.5) 和所选离子跃迁的相对丰度的重复性，仅在三重四极杆 (CV% < 15) 的情况下考虑，已确认适合用于确保明确识别人尿中所有目标分析物的目的。通过对含有莫利司他、罗沙司他或达普司他的尿液样品的分析，验证了新开发方法的适用性。开发的程序能够检测正在研究的化合物及其主要代谢物。